The present invention relates to novel vaccine compositions for parenteral administration, methods for their use and to processes for their preparation. Bacterial and viral diseases of sheep, such as Clostridial diseases, cause considerable economic damage in the agriculture industry. Vaccination is therefore a very important means of controlling these diseases.
Many currently available vaccines are comprised of killed antigens, whether inactivated bacterial cells, viral particles or cellular components, absorbed onto alkali earth metal salts (i.e. aluminium phosphate or aluminium hydroxide) or as water in oil emulsions. For these vaccines it is recommended that naive animals (i.e. animals which have not been previously vaccinated) are treated in a two stage dose regime consisting of an initial dose and a second booster dose several weeks later. The action of the booster dose raises the antibody titre to a level that may sustain protection from a disease causing challenge organism for an extended period. Animals undergoing this vaccination program are usually mustered each year for revaccination. Clearly such initial two-dose administration is time consuming and expensive and is therefore undesirable.
The aluminium based vaccines have been found to have relatively short duration of protection while water-in-oil emulsion vaccines have a longer duration of protection but have been found to be unsuitable for use because they cause unacceptable lesions at the injection sites of the animals (xe2x80x98Experimental Clostridial Oil Emulsion Vaccinesxe2x80x99 Thomson R O. and Batty I., Bull. Off. int Epiz. 1967 67 (11-12)1569-1581; xe2x80x98The Immunogenicity of a multicomponent Clostridial Oil Emulsion Vaccine in sheepxe2x80x99 Thomson et al The Veterinary Record, Jul. 26, 1969). In 1976 Jansen et al. reported the immune response of Cl. botulinum C and D toxoids in a water-in-oil emulsion vaccine and noted that the two-stage aluminium based vaccine was not boosted by the second dose to the same extent as the water in oil compositions (Jansen, B C, Knoetze, P C and Visser, F; Onderstepoort J Vet Res, 43(4) 165-174 (1976)). However, the water in oil compositions gave an undesirable granulomatous swelling resulting from subcutaneous injection of the vaccine in a large percentage of animals which is a severe disadvantage for the vaccine""s commercial use.
WO91/00106 discloses multi-phase emulsions suitable for administering active substances or antigens by injection of the water in oil in water type. These emulsions are produced from pharmaceutically acceptable emulsifiers which when dissolved in an injectable oil, form a homogeneous clear phase and have inversion points approaching the temperature of human or animal bodies. The oils contained in the emulsions include mineral, vegetable or animal oils, and synthetic hydrocarbons. It was observed that these vaccines were well tolerated in pigs and did not cause any local reactions, abscesses or necroses. However, no data were provided regarding the level and duration of the immune responses.
The applicants provide vaccines which are suitable for the prevention of clostridial diseases of sheep and in particular lambs, providing an effective immunity for up to a year or more following a single injection or dose. Therefore, the present invention addresses the problems associated with known vaccines, providing a level of effective immune response in sheep for the period of approximately one year or more following a single injection or dose of vaccine. The present invention also provides vaccines which provide an effective immunity against each of a number of diseases for up to a year or more following a single injection or dose of a multivalent vaccine. The selection of an adjuvant which enhances the antigenic response to clostridial antigens in sheep is thus a problem addressed by the invention. The selection of an adjuvant which enhances the antigenic response to each of a number of micro-organisms in sheep is a particular problem addressed by the invention.
Thus according to the present invention there is provided a sheep vaccine composition comprising:
a) an oily adjuvant acceptable for veterinary purposes comprising:
i) a white mineral oil having a molecular weight of about 250 to 300 and
ii) a mannitol oleate emulsifier and
b) an aqueous phase comprising one or more clostridial antigens.
When used herein the term xe2x80x9csheepxe2x80x9d refers to lambs as well as developing and mature sheep. The vaccines of the invention are particularly useful in the vaccination of lambs.
The vaccine composition is an injectable emulsion of the water in oil type and preferably has a viscosity of about 200 mPas or less, more preferably about 100 mPas to about 150 mPas. The white mineral oil is preferably between about 50% and about 70% by weight of the emulsion more preferably between about 53% and about 63% by weight of the emulsion. The mannitol oleate emulsifier is preferably between about 2% and about 10% by volume of the emulsion more preferably between about 3% and about 7%.
The white mineral oil has a molecular weight of about 250 to 300, preferably about 270 to 290, more preferably about 280. The oil is preferably one which is liquid at 4xc2x0 C. and has a viscosity lower than 100 mPas at 25xc2x0 C. It preferably has a density at 20xc2x0 C. of about 815 to 840 kg/m3, more preferably about 817 to 837 kg/m3. The dynamic viscosity of the oil at 25xc2x0 C. is preferably about 5 to 15 mPas, more preferably about 6 to 13 mPas. The oil preferably has a kinematic viscosity at 40xc2x0 C. of about 5 to 10 mm2/s, more preferably about 7.5 mm2/s. Preferred embodiments of the invention include the commercially available oil Marcol 52 which is supplied by ESSO.
The mannitol oleate emulsifier is preferably an anhydromannitol ether octadecanoate. Preferred emulsifiers have a viscosity at 25xc2x0 C. of about 300 to 400cP, more preferably about 340 to about 360 cP, particularly preferred embodiments are those in which the emulsifier has a viscosity of about 350 cP. The emulsifier preferably has a specific gravity at 20xc2x0 C. of about 0.8 to 1.0, more preferably of about 0.95 to about 0.99, particularly suitable are those with a specific gravity at 20xc2x0 C. of about 0.97. Particularly preferred emulsifiers are those with a refractive index at 25xc2x0 C. of about 1.4 to 1.5, more preferably of about 1.47 to 1.48, particularly those with a refractive index at 25xc2x0 C. of about 1.4748 to 1.4758. Particularly preferred oils are the commercially availably ones Montanide 80, Montanide 103 and Montanide 888 supplied by SEPPIC SA, 75 Quai D-Orsay, 75007 Paris. Montanide 103 and Montanide 888 being more preferred and Montanide 888 being most preferred.
It will be apparent to a person of skill in the art that the proportion of oily adjuvant to aqueous phase included in the emulsion can be adjusted to optimise vaccines including particular antigens and for use in particular animals. It can also be modified to optimise vaccines for administration at a particular site.
The site of administration may also affect the efficacy and/or the site reactions caused by the vaccines. It will be apparent to a person of skill in the art that the site of administration can be selected so as to optimise the effects of vaccines including particular antigens and for use in particular animals. The vaccines exemplified herein were found to be efficacious regardless of site of administration, however site reactions from the exemplified vaccines were more numerous when they were administered at the brisket.
Clostridial antigens suitable for use in the compositions of the present invention include Clostridium perfringens type A, B, C and D, Clostridium septicum, Clostridium tetani, Clostridium chauvoei, Clostridium novyi type B, Clostridium sordelli, Clostridium haemolyticum, Clostridium chauvoei and Clostridium botulinum C and D. Suitable antigens include those which are useful in the treatment of diseases such as Lamb dysentery, Pulpy Kidney disease (enterotosemia), Malignant Oedema (blood poisoning), Tetanus, Blackleg disease and Black disease.
Antigens suitable for use in the present invention are any which provide a suitable immune response, e.g. toxoids or anacultures. Suitable antigens include Clostridium perfringens A, B, C and D toxoids; Clostridium novyi B toxoid; Clostridium chauvoei anaculture; Clostridium septicum toxoid, Clostridium tetani toxoid and Clostridium sordelli toxoid.
The vaccine is preferably a multi-valent vaccine, i.e. a vaccine providing protection against a number of different clostridial diseases by incorporating a number of different clostridial antigens e.g. the vaccine may contain any number of antigens selected from the list provided above. It is particularly useful to provide a multivalent vaccine, i.e. one which provide adequate immune response to a number of pathogens to increase the protection provided by the vaccine. It is particularly difficult to provide multivalent vaccines because it is necessary to provide a vaccine which induces an adequate antigenic response to all the micro-organisms of interest. Thus the threshold antibody responses are described in compendial standards (e.g. Australian Therapeutic Goods order No. 30; British Pharmacopoeia; European Pharmacopoeia and United States Code of Federal regulation). Where compendial standards do not exist (e.g. for Corynebacterium pseudotuberculosis) recognised thresholds based on protection from challenge are accepted.
Preferred embodiments of the invention are vaccines comprising at least two types of clostridial antigen, each one being active against any one of the following: Clostridium perfringens; Clostridium novyi, Clostridium chauvoei; Clostridium septicum and Clostridium tetani. Particularly preferred embodiments being vaccines comprising an antigen to all five diseases listed.
Particular embodiments are vaccines comprising at least two of the following types of clostridial antigen: Clostridium perfringens D toxoid; Clostridium novyi B toxoid; Clostridium chauvoei anaculture; Clostridium septicum toxoid and Clostridium tetani toxoid. A particularly preferred embodiment being a vaccine comprising all five antigens listed.
The vaccine may also comprise antigens against other diseases e.g. Pasteurella antigens such as Pasteurella maltocida and Pasteurella haemolyticum; Corynebacterium antigens such as Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium cystitis and Corynebacterium pilosum; and Haemophilus antigens such as Haemophilus somnus and Haemophilus pleuropneumoniae; Mycoplasma antigens such as Mycoplasma agalactiae and Mycoplasma ovipneumoniae. 
Further preferred embodiments of the invention are those which comprise Corynebacterium antigens such as Corynebacterium pseudotuberculosis, Corynebacterium renale, Corynebacterium cystitis and Corynebacterium pilosum. 
A particularly preferred embodiment of the invention comprises antigens of Clostridium perfringens D toxoid; Clostridium novyi B toxoid; Clostridium chauvoei anaculture; Clostridium septicum toxoid, Clostridium tetani toxoid and Corynebacterium pseudotuberculosis. 
The invention particularly relates to vaccines comprising one or more Clostridial antigens in combination with one or more non-Clostridial antigens.
Co-adjuvants may optionally be included in the vaccines of the present invention. The antigens may be in the form of toxoids or cell antigens but if cell antigens are used a co-adjuvant may be required. Such co-adjuvants may suitably include a saponin (e.g. quil A) or cytokines such as Interleukin- 1, 2, and 4 or muramyl dipeptide. Further emulsifiers such as dioctyl decyl ammonium bromide (DDA) may also be included in the vaccines if desired. The vaccine composition of the present invention may contain one or more antigens and one or more emulsifiers and/or one or more co-adjuvants. Supplements such as selenium which is important for growth and reproductive processes may also be included in the vaccine.
Vaccines according to the present invention may be prepared by dissolving antigens in a suitable aqueous medium such as normal saline, stirring the resultant mixture and adding it to a suitable oil phase. The mixture is then stirred (e.g. at 200 to600 rpm) and/or homogenised (e.g. at 500 to 4500 psi) to the desired viscosity ( less than 200 mPas) and conductivity  less than 0.5 millisiemens at 20xc2x0 C. Preservatives such as thiomersal may optionally be included in the aqueous mixture prior to adding the antigens. This process is preferably carried out at about 20xc2x0 C. to about 25xc2x0 C.
Surprisingly it has been found that the vaccines of the invention can provide a sustained and elevated immune response when administered to the target animals, sheep, in a single dose. They are preferably capable of inducing a response which can be measured, e.g., by ELISA or SN neutralisation titres, for a period of at least 12 months. The vaccine compositions of the present invention are stable and may be stored for several months or even years without loss of antigenic potency. The vaccines are capable of overcoming maternal antibody.
The present invention will now be exemplified with reference to the following Examples by way of illustration only.